With the inevitable depletion of petroleum reserves, fast-growing global populations, and widespread industrialization, there has been an increasing worldwide interest in renewable energies. There is a growing consensus that producing liquid biofuels such as ethanol from renewable and inexpensive lignocellulosic-based plant materials (biomass) has a great potential to meet a large portion of this nation's energy demand in the transportation sector. Moreover, producing biofuels from biomass will simultaneously address three important societal concerns: security of supply (biofuels can be produced locally in sustainable systems), lower greenhouse gas (biofuels recycle carbon dioxide), and support of agriculture. The U.S. Department of Energy (DOE) has set a goal to replace 30% of the liquid transportation fuel with biofuels by 2030.
Similar to ethanol, butanol has many favorable attributes as a fuel molecule. However, it is an underexploited biofuel. Butanol can be produced as a co-product with ethanol and acetone from carbohydrates through fermentation by several solventogenic Clostridia. Compared to the currently popular fuel additive, ethanol, butanol has several advantages. It contains around 22% oxygen which when used as a fuel will result in more complete combustion and low exhaust smoke. In addition, it has a higher energy content (BTU/volume) than ethanol, is more miscible with gasoline and diesel, and has a lower vapor pressure and solubility characteristics which would allow for it to be shipped by pipeline, unlike ethanol.
Solventogenic clostridia are well-known as natural producers of organic solvents via fermentation process. C. acetobutylicum and C. beijerinckii are among the prominent solvent-producing strains capable of producing acetone and butanol as the main fermentation products (Jones, D. T., and D. R. Woods. 1986. Acetone-butanol fermentation revisited. Microbiol. Mol. Biol. Rev. 50:484-524.) Efforts have been made to improve the Clostridia-based butanol fermentation processes by developing new strains and downstream technologies. For example, as described in U.S. Pat. No. 6,358,717, which is incorporated herein by reference in its entirety, Blaschek and others used chemical mutagenesis to develop a mutant strain of Clostridium beijerinckii, BA101 with higher butanol concentration. To circumvent butanol inhibition, Blaschek and others also developed various downstream processes including gas stripping, pervaporation, and liquid-liquid extraction. See, e.g., Ezeji, T. C., Qureshi, N. & Blaschek, H. P. Butanol fermentation research: Upstream and downstream manipulations. Chem Rec 4, 305-314 (2004); US Pat. Pub. No. 2005/0089979; Qureshi et al., Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation, Appl Biochem Biotech 84-6, 225-235 (2000); Formanek et al., Enhanced butanol production by Clostridium beijerinckii BA101 grown in semidefined P2 medium containing 6 percent maltodextrin or glucose. Applied and Env. Microbiol. 63(6):2306-2310 (1997); and Ezeji et al., Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping, Appl Microbiol Biot 63, 653-658 (2004), each of which is incorporated herein by reference in its entirety.
The butanol biosynthesis pathway of the solvent producing Clostridia has been studied, and some of the enzymes involved therein have been purified and characterized. See, e.g., Boynton et al., Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824, Journal of Bacteriology 178, 3015-3024 (1996); Petersen & Bennett, Cloning of the Clostridium acetobutylicum ATCC 824 Acetyl Coenzyme-a Acetyltransferase (Thiolase-Ec 2.3.1.9) Gene, Applied and Environmental Microbiology 57, 2735-2741 (1991); Petersen et al., Molecular-Cloning of an Alcohol (Butanol) Dehydrogenase Gene-Cluster from Clostridium acetobutylicum ATCC-824, Journal of Bacteriology 173, 1831-1834 (1991); and Durre et al., Solventogenic Enzymes of Clostridium acetobutylicum—Catalytic Properties, Genetic Organization, and Transcriptional Regulation, Fems Microbiol Rev 17, 251-262 (1995), each of which is incorporated herein by reference in its entirety.
Butanol fermentation has traditionally been constrained by self-limitation of the reaction due to the toxic effect of the product on the microorganism involved in the process. There is a need for producing solventogenic microorganisms such as clostridia that achieve increased efficiency in the production of bio-butanol.